Nek2 is a mammalian cell cycle-regulated serine/threonine kinase that belongs to the family of proteins related to NIMA of Aspergillus nidulans. Functional studies in diverse species have implicated NIMA-related kinases in G(2)/M progression, chromatin condensation and centrosome regulation. To directly address the requirements for vertebrate Nek2 kinases in these cell cycle processes, we have turned to the biochemically-tractable system provided by Xenopus laevis egg extracts. Following isolation of a Xenopus homologue of Nek2, called X-Nek2B, we found that X-Nek2B abundance and activity remained constant through the first mitotic cycle implying a fundamental difference in Nek2 regulation between embryonic and somatic cell cycles. Removal of X-Nek2B from extracts did not disturb either entry into mitosis or the accompanying condensation of chromosomes providing no support for a requirement for Nek2 in these processes at least in embryonic cells. In contrast, X-Nek2B localized to centrosomes of adult Xenopus cells and was rapidly recruited to the basal body of Xenopus sperm following incubation in egg extracts. Recruitment led to phosphorylation of the X-Nek2B kinase. Most importantly, depletion of X-Nek2B from extracts significantly delayed both the assembly of microtubule asters and the recruitment of gamma-tubulin to the basal body. Hence, these studies demonstrate that X-Nek2B is required for efficient assembly of a functional zygotic centrosome and highlight the possibility of multiple roles for vertebrate Nek2 kinases in the centrosome cycle.
REFERENCES
Arama
E.
, Yanai
A.
, Kilfin
G.
, Bernstein
A.
, Motro
B.
(1998
). Murine NIMA-related kinases are expressed in patterns suggesting distinct functions in gametogenesis and a role in the nervous system.
Oncogene
16
, 1813
–1823
Archer
J.
, Solomon
F.
(1994
). Deconstructing the microtubule-organizing center.
Cell
76
, 589
–591
Barton
A. B.
, Davies
C. J.
, Hutchison
C. A. D.
, Kaback
D. B.
(1992
). Cloning of chromosome I DNA from Saccharomyces cerevisiae: analysis of the FUN52 gene, whose product has homology to protein kinases.
Gene
117
, 137
–140
Boleti
H.
, Karsenti
E.
, Vernos
I.
(1996
). Xklp2, a novel Xenopus centrosomal kinesin-like protein required for centrosome separation during mitosis.
Cell
84
, 49
–59
Brinkley
B. R.
(1985
). Microtubule organizing centers.
Annu. Rev. Cell Biol
1
, 145
–172
Centonze
V. E.
, Borisy
G. G.
(1990
). Nucleation of microtubules from mitotic centrosomes is modulated by a phosphorylated epitope.
J. Cell Sci
95
, 405
–411
Descombes
P.
, Nigg
E. A.
(1998
). The polo-like kinase Plx1 is required for M phase exit and destruction of mitotic regulators in Xenopus egg extracts.
EMBO J
17
, 1328
–1335
Evans
L.
, Mitchison
T.
, Kirschner
M.
(1985
). Influence of the centrosome on the structure of nucleated microtubules.
J. Cell Biol
100
, 1185
–1191
Fava
F.
, Raynaud-Messina
B.
, Leung-Tack
J.
, Mazzolini
L.
, Li
M.
, Guillemot
J. C.
, Cachot
D.
, Tollon
Y.
, Ferrara
P.
, Wright
M.
(1999
). Human 76p: a new member of the-tubulin-associated protein family.
J. Cell Biol
147
, 857
–868
Felix
M.-A.
, Antony
C.
, Wright
M.
, Maro
B.
(1994
). Centrosome assembly in vitro: role of gamma-tubulin recruitment in Xenopus sperm aster formation.
J. Cell Biol
124
, 19
–31
Fry
A. M.
, Schultz
S. J.
, Bartek
J.
, Nigg
E. A.
(1995
). Substrate specificity and cell cycle regulation of the Nek2 protein kinase, a potential human homolog of the mitotic regulator NIMA of Aspergillus nidulans.
J. Biol. Chem
2705
, 12899
–12905
Fry
A. M.
, Nigg
E. A.
(1997
). Characterization of mammalian NIMA-related kinases.
Meth. Enzymol
283
, 270
–282
Fry
A. M.
, Meraldi
P.
, Nigg
E. A.
(1998
). A centrosomal function for the human Nek2 protein kinase, a member of the NIMA-family of cell cycle regulators.
EMBO J
17
, 470
–481
Fry
A. M.
, Mayor
T.
, Meraldi
P.
, Stierhof
Y.-D.
, Tanaka
K.
, Nigg
E. A.
(1998
). C-Nap1, a novel centrosomal coiled-coil protein and candidate substrate of the cell cycle-regulated protein kinase Nek2.
J. Cell Biol
141
, 1563
–1574
Fry
A. M.
, Arnaud
L.
, Nigg
E. A.
(1999
). Activity of the human centrosomal kinase, Nek2, depends upon an unusual leucine zipper dimerization motif.
J. Biol. Chem
274
, 16304
–16310
Glotzer
M.
, Murray
A. W.
, Kirschner
M. W.
(1991
). Cyclin is degraded by the ubiquitin pathway.
Nature
349
, 132
–138
Jones
D. G.
, Rosamond
J.
(1990
). Isolation of a novel protein kinase-encoding gene from yeast by oligodeoxyribonucleotide probing.
Gene
90
, 87
–92
Kellogg
D. R.
, Moritz
M.
, Alberts
B. M.
(1994
). The centrosome and cellular organization.
Annu. Rev. Biochem
63
, 639
–674
Krien
M. J. E.
, Bugg
S. J.
, Palatsides
M.
, Asouline
G.
, Morimyo
M.
, O'Connell
M. J.
(1998
). A NIMA homologue promotes chromatin condensation in fission yeast.
J. Cell Sci
111
, 967
–976
Lu
K. P.
, Means
A. R.
(1994
). Expression of the non-catalytic domain of the NIMA kinase causes a G2arrest in Aspergillus nidulans.
EMBO J
13
, 2103
–2113
Lu
K. P.
, Hunter
T.
(1995
). Evidence for a NIMA-like mitotic pathway in vertebrate cells.
Cell
81
, 413
–424
Martin
O. C.
, Gunawardane
R. N.
, Iwamatsu
A.
, Zheng
Y.
(1998
). Xgrip109: atubulin-associated protein with an essential role in tubulin ring complex (TuRC) assembly and centrosome function.
J. Cell Biol
141
, 675
–687
Merdes
A.
, Ramyar
K.
, Vechio
J. D.
, Cleveland
D. W.
(1996
). A complex of NuMA and cytoplasmic dynein is essential for mitotic spindle assembly.
Cell
87
, 447
–458
Merdes
A.
, Cleveland
D. W.
(1997
). Pathways of spindle pole formation: different mechanisms; conserved components.
J. Cell Biol
138
, 953
–956
Merdes
A.
, Cleveland
D. W.
(1998
). The role of NuMA in the interphase nucleus.
J. Cell Sci
111
, 71
–79
Murray
A. W.
(1991
). Cell cycle extracts.
Meth. Cell Biol
36
, 581
–605
Navara
C. S.
, Hewitson
L. C.
, Simerly
C. R.
, Sutovsky
P.
, Schatten
G.
(1997
). The implications of a paternally derived centrosome during human fertilization: consequences for reproduction and the treatment of male factor infertility.
Am. J. Reprod. Immunol
37
, 39
–49
O'Connell
M. J.
, Norbury
C.
, Nurse
P.
(1994
). Premature chromatin condensation upon accumulation of NIMA.
EMBO J
13
, 4926
–4937
Oakley
B. R.
, Morris
N. R.
(1983
). A mutation in Aspergillus nidulans that blocks the transition from interphase to prophase.
J. Cell Biol
96
, 1155
–1158
Osmani
S. A.
, Pu
R. T.
, Morris
N. R.
(1988
). Mitotic induction and maintenance by overexpression of a G2-specific gene that encodes a potential protein kinase.
Cell
53
, 237
–244
Osmani
A. H.
, McGuire
S. L.
, Osmani
S. A.
(1991
). Parallel activation of the NIMA and p34cdc2 cell cycle-regulated protein kinases is required to initiate mitosis in A. nidulans.
Cell
67
, 283
–291
Osmani
A. H.
, O'Donnell
K.
, Pu
R. T.
, Osmani
S. A.
(1991
). Activation of the nimA protein kinase plays a unique role during mitosis that cannot be bypassed by absence of the bimE checkpoint.
EMBO J
10
, 2669
–2679
Pu
R. T.
, Xu
G.
, Wu
L.
, Vierula
J.
, O'Donnell
K.
, Ye
X. S.
, Osmani
S. A.
(1995
). Isolation of a functional homolog of the cell cycle-specific NIMA protein kinase of Aspergillus nidulans and functional analysis of conserved residues.
J. Biol. Chem
270
, 18110
–18116
Rhee
K.
, Wolgemuth
D. J.
(1997
). The NIMA-related kinase 2, Nek2, is expressed in specific stages of the meiotic cell cycle and associates with meiotic chromosomes.
Development
124
, 2167
–2177
Sawin
K. E.
, Mitchison
T. J.
(1991
). Mitotic spindle assembly by two different pathways in vitro.
J. Cell Biol
112
, 925
–940
Schatten
G.
(1994
). The centrosome and its mode of inheritance: the reduction of the centrosome during gametogenesis and its restoration during fertilization.
Dev. Biol
165
, 299
–335
Schnackenberg
B. J.
, Khodjakov
A.
, Reider
C. L.
, Palazzo
R. E.
(1998
). The disassembly and reassembly of functional centrosomes in vitro.
Proc. Nat. Acad. Sci. USA
95
, 9295
–9300
Schultz
S. J.
, Fry
A. M.
, Sutterlin
C.
, Ried
T.
, Nigg
E. A.
(1994
). Cell cycle-dependent expression of Nek2, a novel human protein kinase related to the NIMA mitotic regulator of Aspergillus nidulans.
Cell Growth Differ
5
, 625
–635
Schweitzer
B.
, Philippsen
P.
(1992
). NPK1, a nonessential protein kinase gene in Saccharomyces cerevisiae with similarity to Aspergillus nidulans nimA.
Mol. Gen. Genet
234
, 164
–167
Simerly
C.
, Zoran
S. S.
, Payne
C.
, Dominko
T.
, Sutovsky
P.
, Navarra
C. S.
, Salisbury
J. L.
, Schatten
G.
(1999
). Biparental inheritance of-tubulin during human fertilization: molecular reconstitution of functional zygotic centrosomes in inseminated human oocytes and in cell-free extracts nucleated by human sperm.
Mol. Biol. Cell
10
, 2955
–2969
Smith
J. C.
, Tata
J. R.
(1991
). Xenopus cell lines.
Meth. Cell Biol
36
, 635
–654
Stearns
T.
, Evans
L.
, Kirschner
M.
(1991
). -Tubulin is a highly conserved component of the centrosome.
Cell
65
, 825
–836
Stearns
T.
, Kirschner
M.
(1994
). In vitro reconstitution of centrosome assembly and function: the central role of-tubulin.
Cell
76
, 623
–637
Tanaka
K.
, Parvinen
M.
, Nigg
E. A.
(1997
). The in vivo expression pattern of mouse Nek2, a NIMA-related kinase, indicates a role in both mitosis and meiosis.
Exp. Cell Res
237
, 264
–274
Tassin
A.-M.
, Celati
C.
, Moudjou
M.
, Bornens
M.
(1998
). Characterization of the human homologue of the yeast Spc98p and its association with-tubulin.
J. Cell Biol
141
, 689
–701
Uto
K.
, Nakajo
N.
, Sagata
N.
(1999
). Two structural variants of Nek2 kinase, termed Nek2A and Nek2B, are differentially expressed in Xenopus tissues and development.
Dev. Biol
208
, 456
–464
Vandre
D. D.
, Davis
F. M.
, Rao
P. N.
, Borisy
G. G.
(1984
). Phosphoproteins are components of mitotic microtubule organising centers.
Proc. Nat. Acad. Sci. USA
81
, 4439
–4443
Vandre
D. D.
, Davis
F. M.
, Rao
P. N.
, Borisy
G. G.
(1986
). Distribution of cytoskeletal proteins sharing a conserved phosphorylated epitope.
Eur. J. Cell Biol
41
, 72
–81
Wu
L.
, Osmani
S. A.
, Mirabito
P. M.
(1998
). A role for NIMA in the nuclear localization of cyclin B in Aspergillus nidulans.
J. Cell Biol
141
, 1575
–1587
Ye
X. S.
, Fincher
R. R.
, Tang
A.
, O'Donnell
K.
, Osmani
S. A.
(1996
). Two S-phase checkpoint systems, one involving the function of both BIME and Tyr15 phosphorylation of p34cdc2, inhibit NIMA and prevent premature mitosis.
EMBO J
15
, 3599
–3610
Ye
X. S.
, Fincher
R. R.
, Tang
A.
, Osmani
A. H.
, Osmani
S. A.
(1998
). Regulation of the anaphase-promoting complex/cyclosome by bimAAPC3and proteolysis of NIMA.
Mol. Biol. Cell
9
, 3019
–3030
Zheng
Y.
, Wong
M. L.
, Alberts
B.
, Mitchison
T.
(1995
). Nucleation of microtubule assembly by a-tubulin-containing ring complex.
Nature
378
, 578
–583
Zimmerman
W.
, Sparks
C. A.
, Doxsey
S. J.
(1999
). Amorphous no longer: the centrosome comes into focus.
Curr. Opin. Cell Biol
11
, 122
–128
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